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All cells need not only to remove damage from their DNA, but also to be able to replicate DNA containing unrepaired damage. In mammalian cells, the major process by which cells are able to replicate damaged templates is translesion synthesis, the direct synthesis of DNA past altered bases. Crucial to this process is a series of recently discovered DNA polymerases. Most of them belong to a new family of polymerases designated the Y-family, which have conserved sequences in the catalytic N-terminal half of the proteins. These polymerases have different efficiencies and specificities in vitro depending on the type of damage in the template.One of them, DNA polymerase eta, is defective in xeroderma pigmentosum variants, and overwhelming evidence suggests that this is the polymerase that carries out translesion synthesis past UV-induced cyclobutane pyrimidine dimers in vivo. DNA polymerase eta is localised in replication factories during DNA replication and accumulates at sites of stalled replication forks. Many studies have been carried out on the properties of the other polymerases in vitro, but there is as yet very little evidence for their specific roles in vivo.

The Restriction Site Mutation (RSM) procedure is a DNA-based method for detecting mutations at any unselected locus. Mutations are identified as alterations of the DNA sequence at a chosen restriction site. DNA from cells exposed to mutagenic treatment is exhaustively digested with the restriction enzyme (RE). Sequences containing the mutated target site are specifically amplified using the polymerase chain reaction (PCR), whereas DNA without mutations at this site will have been cleaved and can not therefore provide a substrate for PCR. We have developed this procedure using both bacterial and mammalian cells. With bacteria, in plasmid reconstruction experiments we were able to detect mutations at a frequency of 10(-6) at an EcoRI site in the AraA locus of Salmonella typhimurium. The detection limit with an RsaI site in the lacI gene of Escherichia coli was 10(-5), and we were able to detect DNA damage and repair after treatment with N-methyl-N-nitrosourea (MNU). With mammalian cells, we have detected mutations induced by ethyl methanesulphonate (EMS) at a TaqI site in the aprt gene of Chinese hamster cells. In extensive studies with normal and repair-deficient human cells, we have detected and sequenced mutations induced by UV-C or UV-B in fibroblasts and lymphoblastoid cells from repair-deficient xeroderma pigmentosum (XP) donors. Similar results were obtained at TaqI sites in three genes, hprt, c-Ha-rasI and p53. These results demonstrate that the system is able to detect and analyse mutations induced at high frequencies. In our extensive attempts to extend the work to conditions of lower mutation frequencies, we have encountered several obstacles, the most serious being false-positive mutant DNA in totally untreated cells. This appeared to be a cell-line specific phenomenon, which we have not been able to eliminate by altering conditions. We propose therefore that, at present, RSM is a suitable method for studying high mutation frequencies at different loci and could be used for mutagen testing with repair-deficient cells. As yet, however, its sensitivity and specificity is not sufficient for population monitoring.

The repair of X-ray-induced DNA damage during G2 cell-cycle phase has been examined in lines of skin fibroblasts from three patients with trichothiodystrophy (TTD), one with apparently normal and two with defective nucleotide excision repair (NER). These responses are compared with those of five lines from clinically normal controls, lines from xeroderma pigmentosum (XP), Cockayne syndrome (CS), Down syndrome (DS), and ataxia telangiectasia (AT) patients. Chromosomal DNA repair was measured as the chromatid aberration frequency (CAF) or total number of chromatid breaks and long gaps per 100 metaphase cells, determined 0.5-1.5 h after X-irradiation (53 rad). Chromatid breaks and gaps (as defined herein) represent unrepaired DNA strand breaks. Only one of the TTD lines, TTD 1BR, showed an abnormally high CAF. This line was shown subsequently to be of a different complementation group, representing a new nucleotide excision repair gene. An abnormally high CAF was also observed, as reported previously, in XP-C, AT and DS but not in CS skin fibroblasts. In addition, cell lines were examined for DNA incision activity by an indirect method in which chromatid aberrations were enumerated with or without ara-C, an inhibitor of repair synthesis, added after X-irradiation. All TTD lines had abnormally low incision activity.

In several patients with the rare hereditary disorder trichothiodystrophy (TTD), a DNA repair defect has been shown to be in the same gene as in xeroderma pigmentosum complementation group D (XP-D). The ERCC-2 gene (excision repair cross-complementing rodent repair deficiency of group 2) has recently been identified as a strong candidate gene for XP-D, since it restores normal UV sensitivity to XP-D cells after transfection. Using Southern blotting, we have analysed the ERCC-2 gene in DNA samples from 28 members of nine Italian families with individuals affected by XP-D (three patients) or by TTD with photosensitivity due to the XP-D defect (eight patients). No major modifications of the ERCC-2 gene were detected with two cDNA probes in either XP-D or TTD patients indicating that the association between TTD and XP-D is not likely to result from a large deletion or rearrangement involving this gene. We found two RFLPs after digestion of the DNA samples with TaqI or MspI, but neither of them could be related to the molecular alteration determining the pathological phenotype. We also analysed a human homologue detected with the hamster sequence isolated by Arrand et al. (1989), which specifically, but partially, complements the DNA repair deficiency in XP-D cells. Our analysis demonstrated that this gene is not the primary gene defective in XP-D. In fact two RFLPs detected with a genomic probe do not co-segregate with the disease in an XP-D family.

Circulating lymphocytes from patients with the DNA-repair-deficient disorders, xeroderma pigmentosum (XP) and ataxia telangiectasia (A-T) have elevated frequencies of mutants at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus. We have analysed the DNA sequence of the hprt gene in mutants from normal donors, and compared them with mutants from XP and A-T individuals. In normal donors we found a range of mutations including principally transitions (40%), transversions (32%) and small deletions (20%). In an excision-deficient XP donor from complementation group C the mutation spectrum was similar to that from normal donors, whereas in an XP variant there was a significantly higher frequency (44%) of small deletions. In the two A-T donors, there was a high frequency of large deletions (22 and 75%) compared with only 4% in normal donors.

Mutants of the fission yeast Schizosaccharomyces pombe which are sensitive to UV and/or gamma-irradiation have been assigned to 23 complementation groups, which can be assigned to three phenotypic groups. We have cloned genes which correct the deficiency in mutants corresponding to 12 of the complementation groups. Three genes in the excision-repair pathway have a high degree of sequence conservation with excision-repair genes from the evolutionarily distant budding yeast Saccharomyces cerevisiae. In contrast, those genes in the recombination repair pathway which have been characterised so far, show little homology with any previously characterised genes.

We have used the SV40-based shuttle vector pZ189 to determine ultraviolet mutation spectra in SV40-transformed cell lines from two patients with Cockayne's syndrome (CS) and ataxia telangiectasia (AT). The shuttle vector was UV-irradiated, transfected into the cells and recovered two days later, after many rounds of replication had occurred. Plasmid DNA was used to transform indicator bacteria in which plasmids containing a mutation in the supF gene resulted in white colonies. Mutant plasmids were analysed both by agarose gels and by DNA sequencing. In contrast to published spectra for xeroderma pigmentosum cells, the types of mutation induced by UV mutation in the CS and AT cell lines were similar to each other and to published spectra for normal cell lines. There were however, some differences in the sequence distribution of the mutations.

Trichothiodystrophy is a genetic disease which in the majority of cases studied is associated with a deficiency in the ability to repair UV damage in cellular DNA. Three categories of UV response have been identified. In type 1 the response is completely normal, whereas type 2 cells are deficient in excision-repair, with properties indistinguishable from those of XP complementation group D. Type 3 cells have normal survival following UV-irradiation and normal rates of removal of cyclobutane pyrimidine dimer sites. Nevertheless repair synthesis is reduced by 50% in these cell strains and this is associated with a marked reduction in the repair of 6-4 photoproducts from cellular DNA. The present results show that 50% or more of repair synthesis at early times after irradiation of normal primary human fibroblasts is attributable to repair of 6-4 products. They also suggest that repair of cyclobutane dimers is crucial for cell survival.

The induction of variants of L5178Y mouse-lymphoma cells resistant to 1-beta-D-arabinofuranosylcytosine (ara-C) has been investigated. The spontaneous mutation frequency was low, around 3 X 10(-8). Resistant variants were induced in a dose-dependent fashion following treatment with ethyl methanesulphonate (EMS), ethidium bromide and 2-acetylaminofluorene, but not with gamma-irradiation. With EMS as mutagen the frequency of ara-C-resistant variants was much lower than that obtained using other selective agents, whereas it was higher with the mutagens acetylaminofluorene and ethidium bromide. Out of 42 resistant clones examined, 38 showed normal sensitivity to thymidine and were destignated as Class 1, the remaining four (Class 2) were usually resistant to thymidine. Cells in Class 1 did not accumulate detectable amounts of ara-C and had negligible deoxycytidine kinase activity. They were about 3000-fold more resistant to the toxic effects of ara-C than were wild-type cells. Cells in Class 2 had a reduced ability to accumulate are-C and had 50% of the deoxycytidine kinase activity of wild-type cells. They were only 20 times more resistant to ara-c than were wild-type cells. The enzymatic defect in these cells is obscure but the phenotypic properties probably result from subtle alterations in the pool of deoxycytidine nucleotides.

An SV40-transformed Fanconi's anaemia (FA) cell line, GM6914, exhibits approximately 2.4-fold increased sensitivity to the cytotoxic effects of nitrogen mustard (NM) when compared with the normal line, MRC5-V1. Host cell reactivation of NM-treated plasmid has been investigated using transient expression vectors which contain the chloramphenicol acetyltransferase (CAT) gene. In both cell types there is a similar, dose-dependent reduction in CAT expression which correlates with an increase in NM-induced DNA-interstrand crosslinking. The data are consistent with two possible mechanisms for inactivation of the plasmid. Either a single crosslink anywhere within the plasmid is sufficient to prevent transcription of the cat gene. Alternatively, inactivation may result from some other more prevalent NM-induced lesions within the cat coding sequence.

Cockayne syndrome (CS) is a genetic disorder showing cellular sensitivity to the lethal effects of UV-irradiation. No defects in unscheduled DNA synthesis or in daughter-strand repair have been detected after UV-irradiation of CS cells. We have studied several aspects of excision repair, particularly at early times after UV-irradiation, and with one exception, we have not been able to detect any difference in the response of normal and CS cells to UV-irradiation, by measuring: (1) the rate of formation of incision breaks in the presence of 1-beta-D-arabinofuranosylcytosine (araC); (2) the amount of repair replication as measured by equilibrium centrifugation; (3) the ligation of repaired DNA to pre-existing DNA; (4) the digestibility of repaired DNA after treatment of nuclei with micrococcal nuclease. The single exception was a pair of CS strains from sibling donors in which the rate of uncoupled incision due to the presence of either araC or the specific inhibitor of DNA polymerase alpha, aphidicolin, was slightly faster than in other cells studied. This effect was absent in the heterozygous parents. However, since this was not seen in two other CS strains in the same genetic complementation group, we can not attribute this increased rate of incision to the defective CS gene. We conclude that, within the limits of resolution of these techniques, CS cells do not have a detectable defect in excision repair.

A comparison has been made, in Chinese hamster cells, of the ability of various methylated oxypurines to inhibit post-replication repair of DNA after UV irradiation and to potentiate UV-induced chromosome aberrations. DNA synthesized in UV-irradiated cells contains gaps, which are subsequently sealed by a process termed post-replication repair. In rodent cells this process is inhibited by caffeine and its analogues. This has been quantitated by measuring the molecular weight of the DNA synthesized in UV-irradiated cells during a 4-h pulse-labelling period in the presence or absence of inhibitors--the lower molecular weight the greater the inhibition. Eight methylated oxypurines were tested; caffeine and chlorocaffeine were always the most potent inhibitors, tetramethyluric acid was inactive, and the other five derivatives (methoxycaffeine, ethoxycaffeine, paraxanthine, theobromine and theophylline) had intermediate effects. Measurements of the potentiation of UV-induced chromosome aberrations showed that treatments with caffeine or chlorocaffeine again had the greatest effects, tetramethyluric acid and also theophylline had no potentiating activity, and methoxycaffeine was intermediate. This correlation between effects at the molecular and cytological levels is consistent with the hypothesis that the inhibition of post-replication repair by methylated oxypurines gives rise to the increased production of chromosome aberrations.

The forward mutation selection system based on resistance to 8-azaguanine has been widely used with cells cultured from a diversity of species and with a variety of mutagens. Ouabain resistance is an alternative selective system. Both systems show a substantial influence of expression time on the number of resistant variants observed after addition of the selective agent such that the frequency reaches a maximum which is dose dependent, and then declines rapidly. Metabolic cooperation has been propsed as the mechanism responsible for this decline with the 8-azaguanine system, but it is less likely to account for the loss of ouabain-resistant variants where it is necessary to invoke generalised effects on the viability of variants due to overcrowding on the plates. A comparison of the two selective systems showed that, with the exception of gamma-irradiation, which was apparently non-mutagenic in the ouabain system, there was broad agreement between the two systems for each mutagen tested. Ethyl methanesulphonate was the most efficient mutagen by a substantial factor. Ouabain resistance permitted greater discrimination particularly between weak mutagens because of the low frequency of spontaneous variants (4 x 10(-7) and also produced data with less intrinsic variability. The absence of gamma ray induced mutation in the ouabain system shows that it may fail to detect certain types of mutagens. Thus the two systems should be used to complement each other. Mutation by the fungicide captan was evaluated using both systems and the positive results indicate that it may pose a hazard to man.

After 16 Jm-2 of UV-irradiation non-dividing normal cells recover normal rates of RNA synthesis within 24 h, whereas in cells from donors with Cockayne syndrome (CS) the rate of RNA synthesis gradually declines. Cultures of a mixed population from 2 CS donors were fused with polyethylene glycol; subsequently they were UV-irradiated and RNA synthesis was measured autoradiographically in mono-, bi-, and multinuclear cells. Genetic complementation was indicated by high levels of RNA synthesis in bi- and multinuclear cells when compared with mononuclear cells. Using this assay, 11 CS strains have been assigned to three complementation groups: 2 into group A, 8 into group B and 1 into group C. The strain in group C is derived from an individual who also had xeroderma pigmentosum (XP), and was the sole known representative of XP-complementation group B.

A rapid procedure for measuring unscheduled DNA synthesis has been studied in detail. Human fibroblasts were brought into the non-dividing state by either growing to confluence or starvation for arginine. Residual semi-conservative synthesis was abolished by hydroxyurea. Hydroxyurea-resistant DNA synthesis which was induced by irradiation and chemical mutagens was presumed to represent repair synthesis and provided a very rapid semi-quantitative procedure for its measurement. Problems were encountered, however, when comparing the quantitative response of different cell strains. The variability between experiments was quite large, and we found that the level of repair synthesis depended not only on the mutagen and the genotype of the cell, but also on physiological factors. This led to some anomalous results. The system was able to detect with ease the large defects in UV-induced repair synthesis in fibroblasts from patients with xeroderma pigmentosum (XP) but it would probably not easily detect less than a 50% reduction in the level of repair synthesis. By extension of this procedure, in combination with cell fusion induced by polyethylene glycol, we have developed a method for carrying out genetic complementation of XP fibroblasts, which does not entail the use of either Sendai virus or of autoradiography. Results of complementation analysis of 4 XP cell strains are presented.

The cell line E2 is a SV40-transformed human fibroblast cell line containing a single integrated copy of the bacterial guanine phosphoribosyl transferase (gpt) gene. Treatment of E2 with ultraviolet light (UV) or ethyl methanesulphonate (EMS) induced the formation of Gpt- derivatives. Several induced derivatives have been isolated, and the structure, expression and revertibility of the gpt gene have been analysed. In the majority of cases the Gpt- phenotype resulted from switching off the gpt gene, in most instances by methylation, but in a few cases by phenotypic switching. Thus mutagenic treatment can result in the inactivation of gene expression in human cells. In a small proportion of Gpt- derivatives the gpt sequences were deleted.